| Lilium regale Wilson, a perennial ornamental crop belonging to the Liliaceae, has great ornamental value. It was an important parent material in breeding programs with good characteristics, such as drought resistance, salt tolerance, diseases resistance.In order to accelerate the resistance breeding of lily, Molecular Breeding has become the hot spot. As lily genome being huge and complicated, there is a lack of functional analysis of gene in lily and even a suitable reference gene for lilium. So isolation of Actin gene from Lilium regale Wilson as the reference gene has important significance on gene expression and functional analysis in lily. Isolation of stress-tolerant related gene and understanding patterns of expressed genes were crucial to provide insights into functional analysis of gene in lily and will lead to the increasing stress resistance of lily by using gene modification method.In this study, full-length cDNA sequence of lilyActin and lilyABC/genes were cloned through a Lilium regale Wilson cDNA library by using the rapid amplification of cDNA ends (RACE) method, and the gene expression in difference tissue of the flower, leaf and bulb were analyzed by the real-time PCR.Sequence analysis and functional prediction of Actin and ABC1genes were based on Bioinformatics. As the accuracy of Real time-PCR mainly relies on the selection of proper reference genes,we performed the gene stability analysis of3candidate reference genes among samples including difference tissues in order to get the most proper reference gene.Furthermore, the expression patterns of6genes in different flower buds,leaf and bulb were assessed by real-time PCR in order to get flower specific expression gene.The main results are as follows:1.Submitted the lilyActin sequence to Genbank and an accession Number JX826390was obtained.The full-length cDNA sequence designated as lilyActin from Lilium regale Wilson was1367bp in length, contains a1134bp open reading frame (ORF) encoding a377amino acid proteins, with91bp in the5’UTR and233bp in the3’UTR. The phylogenetic tree reconstructed on the base of amino acid sequences suggests that the relationship of actin between Lilium regale and Lulipa gesneriana is most intimate. Real time PCR analysis revealed that lilyActin was constantly expressed in various organs of Lilium regale Wilson such as flowers, leaves, bulb.2.Lily reference gene was selected among lilyActin, actin and actin-like mRNA, and the result showed that lilyActin was constantly expressed in various organs,such as flowers, leaves, bulb. The results indicated that lilyActin was the most stable reference gene,while actin was the least stable one.3.The full-length cDNA sequence designated as lilyActin from Lilium regale Wilson was2333bp in length, contains a2007bp open reading frame (ORF) encoding a668amino acid proteins, with262bp in the5’UTR and65bp in the3’UTR. Real time PCR analysis revealed that lilyABCl was all expressed in various organs of Lilium regale Wilson such as flower buds at different developmental stages, leaves, bulb,and the highest expression level was at the leaf. Bioinformatics analysis showed that the gene was localized in the cytoplasm,and there is a highly conserved domain-STYKc, which speculated that the ABC1gene may be associated with the occurrence of cell organism. 4.Six flower specific expression genes(SP, AMDS-boxFT, PIP, PIP-2,14-3-3and WBC-ABC) were obtained from the lily cDNA library while lilyActin was used as the reference gene. These genes exhibited higher expression in flower than leaf and bulb. SP and MADS-boxFT genes exhibited the highest expression level in2cm bud, while PIP gene showed the highest expression in4cm bud, and PIP gene showed the highest expression in6cm bud,14-3-3WBC-ABC genes exhibited the higher expression level in the4cm and10cm bud than the other tissues. |
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